[Risk and features of occupational diseases in nonferrous metallurgy workers of Kolsky Transpolar area].

The study covered data on 977 cases of occupational diseases in 615 workers of nonferrous metallurgy in Kolsky Transpolar area. Findings are high risk of occupational diseases in workers engaged into electrolysis production of aluminium, all nickel reprocessing and pyrometallic copper reprocessing (GR 7.02-10.0). Electrolysis operators and anode operators of aluminium production are more prone to occupational diseases, with bone and muscular disorders (46.8%) prevalent in the morbidity structure. Respiratory diseases are more prevalent (68.2-100%) in the occupational morbidity structure of copper-nickelindustry workers. Conclusion is made on mandatory improvement of the work conditions and more effective individual protective means against occupational hazards in workers of nonferrous metallurgy in Kolsky Transpolar area.

[Influence of industrial pollution of ambient air on health of workers engaged into open air activities in cold conditions].

The article presents the results of a study on assessment of occupational exposure to air pollutants and related health effects in3792 outdoor workers engaged in operations performed in the vicinity of non-ferrous metallurgical facilities in Far North. Findings are that during cold season repeated climate and weather conditions are associated with higher level of chemical hazards and dust in surface air. At the air temperature below -17 degrees C, maximal single concentrations of major pollutants can exceed MAC up to 10 times. With that, transitory disablement morbidity parameters and occupational accidents frequency increase significantly. The workers with long exposure to cooling meteorological factors and air pollution demonstrate significantly increased prevalence of respiratory and circulatory diseases, despite relatively low levels of sculpture dioxide and dust in the air, not exceeding the occupational exposure limits. It has been concluded that severe cold is to be considered asa factor increasing occupational risk at air polluted outdoor worksites dueto more intense air pollution, higher traumatism risk and lower efficiency of filter antidust masks respiratory PPE and due to modification of the toxic effects.

[General and occupational morbidity in workers engaged into electrolysis nickel production in Transpolar Kolsky area].

Findings are that occupational factors in nickel electrolysis workshops induce respiratory and peripheral nervous system diseases. Electrolysis workers demonstrate the highest prevalence and risk of occupational diseases. The authors make a conclusion on necessity to improve prophylactic methods for occupational disorders in these workers.

[Influence of environmental and industrial immunotoxic hazards on clinical course of HIV-infection].

The authors assessed peculiarities of effects caused by the most prevalent environmental pollutants (polychlorinated biphenyls, polycyclic aromatic hydrocarbons, lead, mercury) extensively accumulated in human body on clinical course of HIV-infection.

[Blood heavy metals in women of indigenous ethnic groups in the Far North].

The paper presents the results of an investigation of the blood levels of mercury, lead, and cadmium in women of indigenous ethnic groups in the Far North. A certain correlation was found between the higher incidence of some poor pregnancy outcomes and fetal maldevelopment upon increased maternal exposure to heavy metals. The found correlations were statistically insignificant. There was no significant association of female exposure to heavy metals with menstrual irregularities and the sex ratio of neonates.

[Reduction of a risk of the deleterious effects of persistent toxic substances on the health of the far north population].

The paper presents the general principles and procedure of the development and implementation of measures to decrease and prevent environmental pollution with persistent toxic substances (PTS) in the Russian Arctic and, accordingly, to reduce a risk of the deleterious effects of PTS on human health. Based on the results of a study of PTS in the Russian Arctic (from the Kola Peninsula to Chukotka), the authors first systematized the basic lines and actions and formulated specific measures to reduce the North population's exposure to PTSs, such as polychlorinated biphenyls, organic chlorine pesticides (dichlorodiphenyltrichloroethane, hexachlorocyclohexane, hexachlorobenzene, etc.), and heavy metals (mercury, lead, cadmium). A package of measures is aimed at maximally reducing the presence of PTS-containing objects and materials in the north (via detection, collection, and extermination), at neutralizing the soils in settlement lands, at setting up safe water consumption systems, at organizing effective control over the safe use of chemicals and the levels of PTS in raw food materials and foodstuffs, and at working out recommendations on safe procedures for food purchase, storage, and cooking.

[A priori occupational health risk for workers of ore mining and processing enterprise].

The authors present hygienic evaluation of work conditions, calculate dust loads and risk of stochastic effects in workers of ore mining and processing enterprise. Integral evaluation of the work conditions, according to hygienic criteria, by totality of acting occupational hazards, corresponds to class 3.1-3.4.

[Chemical hair analysis in ore-dressing and processing plant workers].

The article presents results of studies covering elements content of hair and mineral bone density among workers engaged into Utchalinsky ore-dressing and processing enterprise (Utchaly town, Bashkortostan Republic) and among residents of copper-zinc geochemical province. Finding is dysbalance between levels of essential and toxic elements in hair, that is due to environmental and occupational factors. Analysis of elements homeostasis and mineral bone density could be recommended for early diagnosis of osteoporosis.

[Kolsky register of births as an instrument of epidemiologic studies of female reproductive health].

The authors evaluate possible use of Monchegorsk retrospective birth database for demographic and epidemiologic studies of various female reproductive health parameters, newborn health parameters, as well as their connections with occupational and other environmental factors.

Cross-linking studies of steroidogenic electron transfer: covalent complex of adrenodoxin reductase with adrenodoxin.

Bifunctional reagents 3,3'-dithiobis(succinimidyl propionate), 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide and N-succinimidyl 3-(2-pyridyldithio)propionate have been used in an attempt to study molecular organization and covalent cross-linking of adrenodoxin reductase with adrenodoxin, the components of steroidogenic electron transfer system in bovine adrenocortical mitochondria. There was no cross-linking of individual proteins by the bifunctional reagents used, except for adrenodoxin cross-linking with water-soluble carbodiimide. Substantial cross-linking of adrenodoxin reductase with adrenodoxin was observed when water-soluble carbodiimide was used as cross-linking reagent. However, the cross-linked complex failed to transfer electrons. Significant amounts of the functional cross-linked complex (up to 42%) were observed when the proteins were cross-linked with N-succinimidyl 3-(2-pyridyldithio)propionate. Using gel filtration, ion-exchange chromatography and affinity chromatography on adrenodoxin-Sepharose, the complex was obtained in a highly purified form. In the presence of cytochrome P-450scc or cytochrome c, the cross-linked complex of adrenodoxin reductase with adrenodoxin was active in electron transfer from NADPH to heme proteins. The data obtained indicate that there are distinct binding sites on the adrenodoxin molecule responsible for the adrenodoxin reductase and cytochrome P-450scc binding, which suggests that steroidogenic electron transfer may be realized in an organized complex.

Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin.

Selective chemical modification of cytochrome P-450SCC has been carried out with lysine-modifying reagents. Modification of cytochrome P-450SCC with succinic anhydride was shown to result in loss of its ability to interact with intermediate electron transfer protein - adrenodoxin. To identify amino acid residues involved in charge-ion pairing with complementary carboxyl groups of adrenodoxin, cytochrome P-450SCC complex with adrenodoxin was modified with succinic anhydride. Adrenodoxin was then removed and cytochrome P-450 was additionally modified with isotopically labelled reagent. Subsequent chymotryptic hydrolysis of [14C]succinylated cytochrome P-450SCC and separation of digest obtained by combining various types of HPLC resulted in seven major radioactive peptides. The amino acid sequence of the peptides was determined by microsequencing. The major amino groups modified with radioactive succinic anhydride were found to be at Lys-73, -109, -110, -126, -145, -148 and -154 in the N-terminal sequence of cytochrome P-450SCC molecule and at Lys-267, -270, -338 and -342 in the C-terminal sequence. The role of electrostatic interactions in fixation of cytochrome P-450SCC complex with adrenodoxin is discussed.

Chemical modification of adrenocortical cytochrome P-450scc with tetranitromethane.

Selective chemical modification of adrenocortical cytochrome P-450scc, responsible for key stages of steroid biogenesis, with tetranitromethane has been carried out. Nitration of the cytochrome P-450scc tyrosine residues results in heme protein inactivation with syncatalytic loss of enzyme activity. Analysis of the cytochrome P-450scc inactivation kinetics indicates that there are several pools of tyrosine residues, differing in their accessibility to tetranitromethane. The modification of cytochrome P-450scc results in changes in the hemeprotein spectral properties and its conformation which indicates to the involvement of essential tyrosine residue(s) in the heme-protein interaction. Cholesterol and adrenodoxin (high-spin effectors) prevent the inactivation of cytochrome P-450scc with tetranitromethane, i.e., protect the essential tyrosine residue(s) from modification. Possible functions of the tyrosine residues in the cytochrome P-450scc molecule are discussed.

The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria.

A homogeneous cytochrome P-450scc preparation with a specific enzyme content of 18 nmol/1 mg protein has been obtained using affinity chromatography on adrenodoxin-Sepharose under optimal conditions of the protein adsorption onto and desorption from the affinity sorbent. The data on the N-terminal amino acid sequence of the enzyme, along with the results of electrophoretic and spectrophotometric analyses favoured the multistage cholesterol transformation to pregnenolone to be catalyzed by single species of cytochrome P-450scc consisting of one polypeptide chain. Limited proteolysis of cytochrome P-450scc with trypsin resulted, at the initial stages, in the formation (in an equimolar ratio) of two large polypeptide fragments, I and II, with Mr 27000 and 22000, respectively. Prolonged action of trypsin led to the digestion of fragment II and the formation of a stoichiometric amount of fragment III, Mr of about 14000. Cytochrome P-450scc converted by trypsin into equimolar mixtures of fragments I and II or I and III retained the major spectral and functional properties of the native protein. The aspartyl-prolyl linkages, sulphhydryl groups, and surface tyrosine residues are distributed nonuniformly among fragments I and II. These data, as well as a different resistance of the fragments to the action of trypsin, suggest that cytochrome P-450scc consists of two independently folded domains linked with a short loop of the polypeptide chain, the domains being rigidly associated under neutral conditions.

Cross-linking studies of adrenocortical cytochrome P-450scc. Evidence for a covalent complex with adrenodoxin and localization of the adrenodoxin-binding domain.

A cleavable cross-linking reagent, dimethyl-3,3'-dithiobispropionimidate, was used to study the molecular organization of adrenocortical cytochrome P-450scc. Extensive cross-linking was found to occur, resulting in the formation of heterologous oligomers up to octamer. The covalently cross-linked complex of adrenocortical cytochrome P-450scc with adrenodoxin has been obtained by using dimethyl-3,3'-dithiobispropionimidate. In the presence of NADPH and adrenodoxin reductase, electron transfer to cytochrome P-450scc occurs in the complex, and, in the presence of cholesterol, the latter effectively oxidizes to pregnenolone. By using covalently immobilized adrenodoxin and heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate, the adrenodoxin-binding site was shown to be located in the heme-containing, catalytic domain of cytochrome P-450scc. The data obtained indicate the existence of two different sites on the adrenodoxin molecule that are responsible for the interaction with adrenodoxin reductase and cytochrome P-450scc. This is consistent with the model mechanism of electron transfer in the organized complex.

Cross-linking studies of the cholesterol hydroxylation system from bovine adrenocortical mitochondria.

Cytochrome P-450SCC and adrenodoxin were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The sample containing 94% of cross-linked complex and 6% of free cytochrome P-450SCC was obtained after purification on cholate-Sepharose. Cytochrome P-450SCC in cross-linked complex completely preserves its high-spin form in the presence of Tween 20 or pregnenolone. Utilization of radioactively labelled adrenodoxin, chemical cleavage of cytochrome P-450SCC from cross-linked complex with o-iodosobenzoic acid and HPLC for separation of peptides allow us to conclude that the complex of cytochrome P-450SCC with adrenodoxin was cross-linked through two amino acid sequences of cytochrome P-450SCC-Leu-88-Thr-107 and Leu-368-Gly-416. The cross-linked complex of adrenodoxin reductase, adrenodoxin and cytochrome P-450SCC with an apparent molecular mass of 114 kDa was obtained with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. The composition of cross-linked complex was determined by immunoblotting and by evaluation of radioactivity using preliminary N-ethyl[2,3-14C]maleimide-modified adrenodoxin. From this data it appears that the ternary complex may exist in solution.

Primary structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria and some aspects of its functioning on a structural level.

The primary structure of the cholesterol side-chain cleavage cytochrome P-450 (P-450scc) from bovine adrenocortical mitochondria has been determined. At the initial stage an exhaustive chymotryptic digestion of carboxymethylated P-450scc was performed, and the amino acid sequence of 66 peptides was determined. At the second stage an investigation of the amino acid sequence of individual fragments I (Mr 29 800) and II (Mr 26 600) of the limited trypsinolysis of P-450scc was carried out. Fragment I was digested with trypsin, Staphylococcus aureus V8 proteinase and thermolysin; fragment II was cleaved with trypsin and S. aureus V8 proteinase. In addition, the amino acid sequence of some CNBr peptides of P-450scc has been investigated. The primary structure of cytochrome P-450scc determined with protein chemistry methods proved the multistage cholesterol transformation to pregnenolone to be catalyzed by a single species of cytochrome P-450scc which consists of 481 amino acids. The results from protein sequencing of P-450scc are in good agreement with those obtained recently from nucleotide sequencing. The localization of peptide bonds cleaved under limited proteolysis of P-450 with trypsin to fragments I and II, I and III (Mr 16 800) is presented. It is shown that the transformation of P-450scc to P-420 is accompanied by the appearance of an additional trypsin-sensitive peptide bond in the N-terminal part of P-450scc.

The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria. Localization of haem group and domains in the polypeptide chain.

Cytochrome P-450scc consists of two domains linked with a short loop of the polypeptide chain; under hydrolysis by trypsin the domains retain their associated state due to rigid noncovalent interactions. A partial separation of the domains by gel-chromatography on Sephadex G-200 with retention of a haem group in domain I has been achieved after incubation of the trypsin-modified cytochrome P-450scc in 50 mM phosphate buffer (pH 7.2)/1 M NaCl/0.3% sodium cholate/0.3% Tween 80. The separation of domains I and II to individual fragments of the haemoprotein polypeptide chain has been achieved by chromatography under denaturation conditions on the activated thiopropyl-Sepharose via a selective covalent immobilization of domain II. Dissociation of a complex of domains I and II has been effectuated in the presence of 7 M guanidine. Structural characteristics of individual domains have been investigated. It is established that domain I containing a haem group is the N-terminal moiety, and domain II, the C-terminal moiety of the polypeptide chain of cytochrome P-450scc. The pathways of limited trypsinolysis of the native cytochrome P-450scc have been determined. The peptides containing cysteine residues localized on the surface of domain II and responsible for the interaction of haemoprotein with activated thiopropyl-Sepharose have been isolated in a homogeneous form and their amino-acid sequences have been assessed.

Chemical modification of rabbit IgG with N-dansylaziridine. Investigation of the properties of dansylated antibodies.

To elucidate the effect of antigen binding fluorescent thiol reagent, N-dansylaziridine (DAZ), which is sensitive to the changes in the microenvironment, was used for modification of rabbit IgG hinge region cysteine residue. DAZ binds to hinge region Cys 226 as evidenced by the structural analysis. Labelling of IgG with DAZ does not alter its conformation, hydrodynamic behavior, nor its antigen binding properties. Upon antigen bindings, the fluorescence intensity of modified IgG increases about 80%. This result indicates that interaction of antibodies with antigen is accompanied by the conformational changes in the IgG hinge region.

Interaction of cytochrome P-450scc with cytochrome b5.

Spectrophotometric, affinity chromatography and cross-linking experiments provided evidence that cytochrome P-450scc from bovine adrenocortical mitochondria forms a tight complex with cytochrome b5 from rabbit liver microsomes. In the reconstituted system cholesterol side chain activity of cytochrome P-450scc was enhanced by the addition of cytochrome b5.

Is cytochrome P-450scc a transmembrane protein?

The topology of cytochrome P-450scc in the inner mitochondrial membrane of adrenal cortex has been investigated using monospecific antibodies to cytochrome P-450scc and its fragments F1 (Ile1-Arg250), F2 (Asn257-Ala481) and F3 (Asn257-Arg399). Antibodies to F1 and F2 were shown to effectively bind to the matrix and cytosolic sides of the inner membrane. Antibodies to F3 specifically interacted only with the matrix side of the membrane. These data are consistent with a model of molecular organization which shows that cytochrome P-450scc is a transmembrane protein, both N- and C-terminal sequences of the cytochrome being able to span the membrane.